Difference between revisions of "Team:Paris Saclay/Notebook/September/19"

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!dCas9 ST - GFP 11 clone 8
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!dCas9 ST - GFP 11 (pPS16_017) clone 8
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!dCas9 ST - GFP 11 clone 8
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!dCas9 ST - GFP 11 (pPS16_017) clone 8
 
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|Primers
 
|Primers
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====Gel of PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8====
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''By Maxence, Mahnaz, Coline & Caroline''
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4 µL of each PCR products and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
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PCR products expected were :
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{| class="wikitable"
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|-
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!PCR products
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!Expected band size (bp)
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|Product 1 obtained by iPS174 & iPS175
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|5837
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|-
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|Product 2 obtained by iPS173 & iPS176
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|1599
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|-
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|}
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GEL
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{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Revision as of 13:55, 19 September 2016

Monday 19th September

Lab work

Visualization

Glycerol stocks of clones 7, 8, 9 and 10 containing FKBP - GFP 10 in pSB1C3 (pPS16_018)

"By Maxence, Mahnaz Coline & Caroline"

The glycerol stock of the bacteria with the following plasmids were made.

  • pPS16_018 (FKBP - GFP 10) clone 7
  • pPS16_018 (FKBP - GFP 10) clone 8
  • pPS16_018 (FKBP - GFP 10) clone 9
  • pPS16_018 (FKBP - GFP 10) clone 10

500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.

Plasmids extraction of clones 7, 8, 9 and 10 containing GFP 1.9 in pSB1C3 (pPS16_018)

"By Maxence, Mahnaz Coline & Caroline"

The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:

  • pPS16_018 (FKBP - GFP 10) clone 7
  • pPS16_018 (FKBP - GFP 10) clone 8
  • pPS16_018 (FKBP - GFP 10) clone 9
  • pPS16_018 (FKBP - GFP 10) clone 10

PCR of pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 to correct mutations

By Maxence, Mahnaz, Coline & Caroline

In order to correct mutations in dCas9 ST - GFP 11, PCR was performed with the following protocol.

For each 50μl of reaction, mix the following reagents :

  • 1 µL of matrix
  • 1 µL of dNTPs (10mM)
  • 2.5 µL of each primer mix (10µM)
  • 10 µL of buffer HF (5X)
  • 0,5 µL of Phusion polymerase
  • 32.5 µL of nuclease free water

Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow:

Step Temperature Time
Initial denaturation 98°C 30sec
30 cycles 98°C 10sec
72°C 30sec
72°C t
Final Extension 72°C 5min
Hold 4°C $\infty$ 
Primers used were:
Matrix dCas9 ST - GFP 11 (pPS16_017) clone 8 dCas9 ST - GFP 11 (pPS16_017) clone 8
Primers iPS174 and iPS175 iPS173 and iPS176
Tm 72°C 72°C
t 3min 50sec

Gel of PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8

By Maxence, Mahnaz, Coline & Caroline

4 µL of each PCR products and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

PCR products Expected band size (bp)
Product 1 obtained by iPS174 & iPS175 5837
Product 2 obtained by iPS173 & iPS176 1599

GEL







Université Paris Sud
91405 Orsay, France
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*This wiki was conceived during long nights of work and a great coffee experience in the beautiful Orsay, France.